Discovery of Ureido-Based Apcin Analogues as Cdc20-specific Inhibitors against Cancer

Cdc20 is a promising drug target that plays an important role in the mid-anaphase process of cellular mitosis, and Apcin is the only reported core structure of the Cdc20-specific inhibitor. Some potent Apcin derivatives were obtained in our previous research, and a structure–activity relationship was determined. In this study, we designed and synthesized a series of ureido-based Apcin derivatives. The proliferation-inhibition experiments on four cancer-cell lines showed that ureido skeleton could promote the anti-proliferation activity of purine-substituted compounds, whereas the ureido analogues with pyrimidine substitutes showed no significant improvement in the inhibitory effect compared with the original ones. Further tests confirmed that ureido-based compounds can enhance the binding affinity to Cdc20 by increasing the levels of Cdc20 downstream proteins. Compound 27 revealed a remarkably antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM) and potent binding affinity to Cdc20. Moreover, compound 20 induced caspase-dependent apoptosis and cell-cycle arrest at the G2/M phase, and compound 27 induced caspase-dependent apoptosis and promoted microtubule polymerization. Finally, a molecular-docking simulation was performed for compounds 20 and 27 to predict the potential ligand–protein interactions with the active sites of the Cdc20 proteins.


Introduction
Anaphase-promoting complex/cycle (APC/C) is a multifunctional ubiquitin protein ligase that ubiquitinates against diverse ubiquitinated substrates to regulate various cellular processes such as cell division, differentiation, genome stability, energy metabolism, cell death, autophagy, and carcinogenesis [1,2]. APC/C works only when two coactivators-Cdc20 and Cdh1-join in. Ubiquitination of substrates by APC/C requires the formation of an APC/C-activator-substrate complex, and Cdc20 and Cdh1 act on APC/C to target specific substrates at different stages of the cell cycle. The WD40 domains of Cdc20 and Cdh1 located at C-terminus provide a binding platform to recruit APC/C substrates [3][4][5][6].
TCGA statistics show that the Cdc20 gene is overexpressed in a variety of human tumor tissues, including breast cancer, cervical cancer, colorectal cancer, liver cancer, and other cancer tissues. Statistical analysis of clinical trials by many researchers provides

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervicalcancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 μM), MCF-7 (IC50 = 0.27 ± 0.06 μM), MDA-MB-231 (IC50 = 0.32 ± 0.04 μM), and Hepg2 (IC50 = 0.24 ± 0.11 μM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously synthesized pyrimidine compound 7d (IC50 = 63.20 ± 0.90μM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).

In Vitro Antiproliferative Assay and Prediction of Lipid Permeability and Toxicity in Silico
As shown in Table 2, the results of the antiproliferative assay in vitro of the target compounds were similar in different cell lines, among which Mcf-7 and Mda-mb-132 are both breast-cancer cells, HepG2 is a hepatocellular carcinoma cell, and Hela is a cervical-cancer cell. Among all compounds, purine derivative 27 revealed a remarkably broad antitumor activity pattern against Hela (IC 50 = 0.06 ± 0.02 µM), MCF-7 (IC 50 = 0.27 ± 0.06 µM), MDA-MB-231 (IC 50 = 0.32 ± 0.04 µM), and Hepg2 (IC 50 = 0.24 ± 0.11 µM), which was better than that of compound 9f obtained in the previous study. Dichloro-substituted pyrimidine derivative 20 showed potent antiproliferative effects against all the tested tumor-cell lines and had stronger proliferation-inhibition activity compared to that of the previously syn- thesized pyrimidine compound 7d (IC 50 = 63.20 ± 0.90 µM, Hela). At the same time, the lower cLogP value of compound 20 compared to compound 7d implies that compound 20 had greater lipid permeability; in addition, the toxicity value of compound 20 was slightly less than that of compound 7d (Table 3).  Among the ureido-based Apcin derivatives, the proliferation-inhibition experiments on four cancer-cell lines showed that the ureido analogues with pyrimidines substitutes had no significant alteration to the original ones (1 vs. Apcin vs. 20 vs. 19), whereas the ureido skeleton could promote the activity of the purine-substituted compound (27 vs. 9f). Compound 9f had greater lipid permeability than compound 27 ( Table 3), suggesting that compound 27 has stronger anti-proliferative activity due to other mechanisms. Compounds with no substituents on pyrimidines were described as R 2 -NH 2 (Scheme 1), including nitroimidazole (1), phenethyl (2), cyclohexyl (3), 2-morpholinoethylamine (4), ethanolamine (5), and benzenesulfonyl (6), all of which showed poor activity (IC 50 > 80 µM). Besides, the substitution on the pyrimidines displayed a strong influence on activity, such as chlorine (9), cyano (15), fluorine (16), which showed dramatically reduced activities versus the most active compound, whereas in the pyrimidine substituted with electron-withdrawing substituents, such as trifluoromethyl (7), meta-dichloro (17), and dichloroamino (20), the antiproliferative activity of the compound was greatly enhanced (IC 50 < 40µM). In addition, the position of the substituents on the pyrimidine also had some effect on the activity. When only the amidopyrimidine counterpoint was replaced, the activity of the compounds was very poor (9, 10, 13, 14, 15). When the amidopyrimidine was substituted in the orthoand meso-positions, the activity of the compounds was significantly enhanced (7, 20, 22-26). Multi-substituted compounds like compounds 20, 25, and 26 showed good activity compared with Apcin.
Under the premise of the ureido-based Apcin derivatives, the activity of 2-fluoroadenine compounds against Hela (27, IC 50 = 0.06 ± 0.02 µM; 28, IC 50 = 0.17 ± 0.08 µM; 29, IC 50 = 0.08 ± 0.01 µM) was much stronger than that of any pyrimidine compounds. Among the purine compounds, the activity of compound 27 (IC 50 = 0.06 ± 0.02 µM, Hela) substituted with 2-morpholinoethylamine in R 3 -NH 2 was stronger than that of phenethylamine-substituted compound 28 (IC 50 = 0.17 ± 0.08 µM). It was speculated that the difference in water solubility might be the reason for the difference in activity of the compounds. * IC 50 = Compound concentration required to inhibit tumor-cell proliferation by 50%. Data are expressed as the mean ± SD from the dose-response curves of at least three independent experiments. # The results for compounds 7b and 7d came from our previous studies [19].
In summary, the data on toxicity (Table 3) showed that most of the compounds may have had hepatotoxicity, reproductive toxicity, and acute oral toxicity, but the acute oral toxicity was relatively small. In addition, a few compounds may have had nephrotoxicity. Ureido-based Apcin analogues exhibited better antiproliferative activity compared to carbamate-based ones. The ureido-based pyrimidine compounds exhibited the most effective antiproliferative activity when metronidazole was substituted with a benzene ring, and the substituent activity was phenethyl > metronidazole > 2-morpholineethanol against Hela. However, morpholine was the preferred structural element of the purine compounds (2-morpholineethanol > metronidazole > phenethyl), which is consistent with our previous findings (9f, IC 50 = 0.3 ± 0.2 µM; 9a, IC 50 > 300 µM; 9b, IC 50 = 51.2 ± 0.9 µM; Hela) [19]. We speculate that it is because the membrane permeability of benzyl-substituted purine compounds was too poor to enter the cell for their efficacy. However, the predicted results of cLogP values (Table 3) were inconsistent with the results of anti-proliferation experiments (28 > 27 > 29, metronidazole > 2-morpholineethanol > phenethyl).
When R 3 -NH 2 was substituted with morpholine, ureido-based purine compound 27 (IC 50 = 0.06 ± 0.02 µM, Hela) showed at least 3000-fold more activity than the Apcin reference compound (IC 50 = 181.88 ± 12.49 µM, Hela). Fluoro-substituted and ureido-based analogues exhibited the most effective antiproliferative activity. Based on the results of the Western blot, as shown below( Figure 1, the level of Cdc20 protein in Hela cells was relatively high, which was selected as the cell line for further experiments. Therefore, we selected pyrimidine compound 20 and purine compound 27 as the compounds with the strongest proliferation-inhibition activity against Hela for subsequent mechanism research. * "+" means toxic, "-"means nontoxic, and the number means probability. Predictive toxicity value was determined using the admetSAR website. ClogP was predicted using the SwissADME website.

Surface Plasmon Resonance (SPR) Assay
Surface plasmon resonance (SPR) assay provides information on the affinity and kinetics of molecular interactions, and the affinity value (Kd) associated with the interaction may be used to investigate the binding efficiency. The results of the SPR experiment are shown in Table 4 and Figure 2. Compounds 20 and 27 exhibited higher binding ability to Cdc20 protein than compounds 7d and 9f.
Interaction of compounds 20 and 27 with Cdc20, along with Apcin acting as the reference compound, were determined by SPR experiments. The results showed that compounds 20 and 27 and Apcin interacted with the human recombinant Cdc20 protein on the chip, with Kd values of 79.6 µM, 97.0 µM, and 236 µM, respectively (as shown in Figure 2 and Table 4). The experimental results prove that the two selected compounds were consistent with Apcin and could be reversibly combined with Cdc20, with obvious binding and dissociation phases. Among them, the binding affinity of compound 20 to Cdc20 (Kd = 79.6 µM) was about three times that of Apcin and Cdc20 (Kd = 236 µM) and was slightly stronger than that of the previous compound 7d. As compound 20 showed potent antiproliferative effects against all the tested tumor-cell lines compared to compound 7d, it is suggested that the binding affinity of compounds 20 and 7d was consistent with its antiproliferative potency.

Surface Plasmon Resonance (SPR) Assay
Surface plasmon resonance (SPR) assay provides information on the affinity and kinetics of molecular interactions, and the affinity value (Kd) associated with the interaction may be used to investigate the binding efficiency. The results of the SPR experiment are shown in Table 4 and Figure 2. Compounds 20 and 27 exhibited higher binding ability to Cdc20 protein than compounds 7d and 9f. Compound 27, a compound representative of purine, showed a stronger affinity with Cdc20 (Kd = 97 µM) than Apcin (Kd = 236 µM), and was higher than the previous compound 9f. Considering the results of purine compound 27 and pyrimidine compound 20, the binding affinity was inconsistent with its antiproliferative potency (Kd value: 20 > 27 > Apcin, IC 50 value: 27 < 20 < Apcin), suggesting that compound 27 may have other anti-cancer mechanisms. The results prove that ureido-based Apcin analogues may be more advantageous than carbamate ones.

Molecular-Docking Simulation
In this study, we simulated the interaction of the compound with the Cdc20 protein (PDB ID: 4n14) using Molecular Operating Environment (MOE 2015) and showed the ligand interactions between compound and protein through the 2D map in Figure 3with the corresponding score. The results of the affinity values showed that ureido-based Apcin analogues had stronger binding ability with Cdc20 than the carbamate ones (20 > 27 > 7d > 9f). and the absolute binding score of compound 20 with Cdc20 protein was greater than that of compound 7d (20 vs. 7d), which proved that compound 20 had stronger binding ability with Cdc20 protein. The docking results are consistent with the results of our SPR experiment. In addition, compared with compound 9f, compound 27 did not show much advantage in molecular docking ( Figure 3D,3E). They only showed two hydrogen bonds with Asp177; however, their absolute binding score with Cdc20 protein shows that compound 27 had stronger binding ability with Cdc20 (27 vs. 9f), which explains the result of the SPR experiment. Due to the restriction of the Cdc20 protein pocket, the ureido was exposed outside the pocket and was unable to form hydrogen bonds with the surrounding amino acids. We suspect that compound 27 had an unknown interaction with Cdc20 protein and may need to be elucidated by eutectic structure with Cdc20 protein. Our next plan is to consider exploring the binding mode of Cdc20 protein and compound 27.
All docking results show that ureido-based Apcin analogues could maintain a similar binding mode as Cdc20 binding with Apcin and had stronger binding ability with Cdc20 than the carbamate ones.  All of the compounds formed hydrogen bonds with backbone atoms from Asp177. The hydrophobic trichloromethyl groups of compounds 20 and 27 were found to be buried in the pocket, which is similar to Apcin. When compound 7d was compared with compound 20, compound 20 increased one hydrogen interaction with Gly214 ( Figure 3B,3C), and the absolute binding score of compound 20 with Cdc20 protein was greater than that of compound 7d (20 vs . 7d), which proved that compound 20 had stronger binding ability with Cdc20 protein. The docking results are consistent with the results of our SPR experiment. In addition, compared with compound 9f, compound 27 did not show much advantage in molecular docking ( Figure 3D,3E). They only showed two hydrogen bonds with Asp177; however, their absolute binding score with Cdc20 protein shows that compound 27 had stronger binding ability with Cdc20 (27 vs . 9f), which explains the result of the SPR experiment. Due to the restriction of the Cdc20 protein pocket, the ureido was exposed outside the pocket and was unable to form hydrogen bonds with the surrounding amino acids. We suspect that compound 27 had an unknown interaction with Cdc20 protein and may need to be elucidated by eutectic structure with Cdc20 protein. Our next plan is to consider exploring the binding mode of Cdc20 protein and compound 27.
All docking results show that ureido-based Apcin analogues could maintain a similar binding mode as Cdc20 binding with Apcin and had stronger binding ability with Cdc20 than the carbamate ones.

Western Blot
The results of the Western blot show that compounds 20 and 27 increased the levels of Cdc20 downstream proteins but had no effect on the Cdc20 protein level, consistent with Apcin (Figures 1 and 4).

Western Blot
The results of the Western blot show that compounds 20 and 27 increased the levels of Cdc20 downstream proteins but had no effect on the Cdc20 protein level, consistent with Apcin (Figures 1 and 4). It is reported that Cdc20 protein is overexpressed in various cancer-cell lines. We selected four cells lines, including Mda-mb-231, Hepg2, Mcf-7, and Hela, to investigate the Cdc20 levels and detect the expression of Cdc20 (Figure 1). The results show that the level of Cdc20 protein in Hela cells was relatively high, and was selected as the cell line for further experiments. Similarly, neither compound 20 nor compound 27 had any effect It is reported that Cdc20 protein is overexpressed in various cancer-cell lines. We selected four cells lines, including Mda-mb-231, Hepg2, Mcf-7, and Hela, to investigate the Cdc20 levels and detect the expression of Cdc20 (Figure 1). The results show that the level of Cdc20 protein in Hela cells was relatively high, and was selected as the cell line for further experiments. Similarly, neither compound 20 nor compound 27 had any effect on the level of Cdc20 protein in Hela cells, as shown in Figure 4, indicating that the compounds did not act by regulating the translational and post-translational modification of Cdc20, consistent with the mechanism of action of a Cdc20-specific inhibitor.
Cyclin B1, securin, and Bim are the key specific downstream substrates of Cdc20, which are related to cell cycle and apoptosis. Cyclin B1 is mainly expressed in the G2/M phase of cells and regulates the cell-cycle progression. Securin protein prevents the proteolysis of the cohesin complex and the subsequent segregation of the chromosomes during mitosis. As shown in Figure 4, after the cells were treated with compound 20 or 27, the level of cyclin B1 and securin protein was significantly increased, which is consistent with Apcin. Cleaved PARP is a 116 a nuclear polymerase, which is a highly conserved ribozyme involved in DNA repair and apoptosis. Bim is a protein in the Bcl-2 family that has pro-apoptotic activity. Both compound 20 and compound 27 could significantly increase the expression of cleaved PARP and Bim, and the effect of compound 27 was more significant, indicating that the pro-apoptotic effect of compound 27 is stronger than that of compound 20. These results reveal that compounds 20 and 27 were consistent with the mechanism of Apcin as Cdc20 inhibitors. At the same time, further experiments are needed to verify whether the compounds have any effect on cell cycle and apoptosis.

The Annexin V-FITC/PI Double-Staining Fluorescence Experiment
The annexin V-FITC/PI double-staining fluorescence experiment of Hela cells was carried out to examine the effect of the compounds on cell apoptosis. As shown in Figures 5 and 6

The Annexin V-FITC/PI Double-Staining Fluorescence Experiment
The annexin V-FITC/PI double-staining fluorescence experiment of Hela cells was carried out to examine the effect of the compounds on cell apoptosis. As shown in Figures  5 and 6 Apoptotic rates were increased in a concentration-dependent way in Hela cells treated with Apcin, compound 20, and compound 27 for 24 h. Compound 20 at 80 μM and compound 27 at 1 μM showed more potency than Apcin at 300 μM. The results are consistent with the trend of cell proliferation-inhibition experiments and Western blot experiments, indicating that the inhibitory effect of the compound on the proliferation of Hela was probably related to its apoptosis-inducing effect.
To test whether the compound blocked mitotic exit in Hela cells, an annexin V-FITC/PI assay was conducted in Hela cells treated with Apcin, compound 20, or compound 27 for 24 h, respectively. A significant increase in the number of cells in the mitotic G2/M phase was found after treatment with Apcin or compound 20 (Figure 6), and the experimental results are concentration-dependent: 34.87% of cells were in the G2/M phase after Apcin (150 μM) treatment, and 41.09% of cells were in the G2/M phase after compound 20 (40 μM) treatment, indicating that compound 20 showed stronger ability to block mitotic exit than Apcin did. After treated with compound 27, the number of cells in the G2/M phase was slightly reduced, but increasing the concentration of 27 had little effect on the cell mitotic exit, which is inconsistent with the results of Apcin. It is suggested that compound 27 may have different mechanisms of pro-apoptotic action than Apcin. To test whether the compound blocked mitotic exit in Hela cells, an annexin V-FITC/PI assay was conducted in Hela cells treated with Apcin, compound 20, or compound 27 for 24 h, respectively. A significant increase in the number of cells in the mitotic G2/M phase was found after treatment with Apcin or compound 20 (Figure 6), and the experimental results are concentration-dependent: 34.87% of cells were in the G2/M phase after Apcin (150 µM) treatment, and 41.09% of cells were in the G2/M phase after compound 20 (40 µM) treatment, indicating that compound 20 showed stronger ability to block mitotic exit than Apcin did. After treated with compound 27, the number of cells in the G2/M phase was slightly reduced, but increasing the concentration of 27 had little effect on the cell mitotic exit, which is inconsistent with the results of Apcin. It is suggested that compound 27 may have different mechanisms of pro-apoptotic action than Apcin.

Microtubule Polymerization-Inhibition Experiment
Our research previously proved that adenine compound 9f had dual inhibitory effects of Cdc20 and microtubules. Compound 27 can also disrupt the polymerization of tubulin with a different mechanism, as shown in Figure 7.
was used as the reference compound to investigate whether compound 27 could disrupt the organization of the cellular microtubule network. A microtubule polymerization-inhibition experiment was carried out in vitro, as shown in Figure 7. Compound 20 at 30 μM slightly promoted microtubule polymerization, but compound 27 at 3 μM and 30 μM was much more effective and promoted microtubule polymerization in a concentrationdependent manner, indicating that compound 27 is a potent promotor of tubulin assembly. Compound 27 and paclitaxel had the same mechanism of action, which could promote microtubule polymerization. Like paclitaxel, compound 27 might prevent the formation of normal mitotic spindles, cause chromosome breakage, and inhibit cell replication, which might be the reason why the cancer-cell proliferation activity of compound 27 was greatly enhanced. Figure 7. The effect of compound 20 and compound 27 on in vitro tubulin polymerization was tested. Polymerization of purified tubulin was performed in a cell-free assay. Tubulin protein was incubated at 37 °C in a reaction buffer exposed to vehicle control or test compounds at the indicated concentrations. Absorbance at 340 nm was monitored at 37 °C every 30 s for 1 h.

Bioled-Egg Model Analysis
We carried out Bioled-egg model analysis using the SwissADME website. The dots in the white ellipse represent compounds that are most likely to be passively absorbed by the gastrointestinal tract. The dots in the yellow represent compounds that are most likely to penetrate the CNS through the BBB. The white and the yolk are not mutually exclusive. Molecules that are not expected to be absorbed well and BBB-permeable molecules are located in the gray area. The results (Figure 8) show that Apcin and compound 27 were substrates of P-GP (drug resistance), and compound 20 was not a substrate of P-GP; it was difficult for the gastrointestinal tract to absorb Apcin, but compounds 20 and 27 were absorbable. None of the three compounds could cross the blood-brain barrier. Figure 7. The effect of compound 20 and compound 27 on in vitro tubulin polymerization was tested. Polymerization of purified tubulin was performed in a cell-free assay. Tubulin protein was incubated at 37 • C in a reaction buffer exposed to vehicle control or test compounds at the indicated concentrations. Absorbance at 340 nm was monitored at 37 • C every 30 s for 1 h.
The structure of compound 27 is similar to that of compound 9f; therefore, paclitaxel was used as the reference compound to investigate whether compound 27 could disrupt the organization of the cellular microtubule network. A microtubule polymerization-inhibition experiment was carried out in vitro, as shown in Figure 7. Compound 20 at 30 µM slightly promoted microtubule polymerization, but compound 27 at 3 µM and 30 µM was much more effective and promoted microtubule polymerization in a concentration-dependent manner, indicating that compound 27 is a potent promotor of tubulin assembly. Compound 27 and paclitaxel had the same mechanism of action, which could promote microtubule polymerization. Like paclitaxel, compound 27 might prevent the formation of normal mitotic spindles, cause chromosome breakage, and inhibit cell replication, which might be the reason why the cancer-cell proliferation activity of compound 27 was greatly enhanced.

Bioled-Egg Model Analysis
We carried out Bioled-egg model analysis using the SwissADME website. The dots in the white ellipse represent compounds that are most likely to be passively absorbed by the gastrointestinal tract. The dots in the yellow represent compounds that are most likely to penetrate the CNS through the BBB. The white and the yolk are not mutually exclusive. Molecules that are not expected to be absorbed well and BBB-permeable molecules are located in the gray area. The results (Figure 8) show that Apcin and compound 27 were substrates of P-GP (drug resistance), and compound 20 was not a substrate of P-GP; it was difficult for the gastrointestinal tract to absorb Apcin, but compounds 20 and 27 were absorbable. None of the three compounds could cross the blood-brain barrier.
In summary, some researchers found that Apcin reduced the proportion of mitotic cells in a dose-dependent manner and shortened the duration of mitosis after nocodazole (microtubule-destabilizing agent) or taxol (microtubule stabilizer) treatment, which was the result of Apcin's specific binding to the D-box pocket of Cdc20 [24]. The Apcin derivatives we synthesized might have similar microtubule-disrupting effects as nocodazole or taxol, which is also the reason for the greatly increased antiproliferation-inhibition activity. It was suggested that another mechanism of action might exist in compound 27; further exploration needs to be done. The blue dots indicate that the molecule is predicted to be a substrate for P-glycoprotein (PGP + ) and will therefore be actively pumped out of the brain or gastrointestinal lumen. If the compound is not to be a substrate of p-glycoprotein (PGP, the relevant points are shown in red. WLOGP is the method for evaluating the lipophilicity of compounds shown in the ordinate diagram, and TPSA is the polar surface-area unit A of the compound. In summary, some researchers found that Apcin reduced the proportion of mitotic cells in a dose-dependent manner and shortened the duration of mitosis after nocodazole (microtubule-destabilizing agent) or taxol (microtubule stabilizer) treatment, which was the result of Apcin's specific binding to the D-box pocket of Cdc20 [24]. The Apcin derivatives we synthesized might have similar microtubule-disrupting effects as nocodazole or taxol, which is also the reason for the greatly increased antiproliferation-inhibition activity. It was suggested that another mechanism of action might exist in compound 27; further exploration needs to be done. Table 4. LogP predictive value (cLogP) and toxicity predictive value of compounds *.

Compounds
Number The blue dots indicate that the molecule is predicted to be a substrate for P-glycoprotein (PGP + ) and will therefore be actively pumped out of the brain or gastrointestinal lumen. If the compound is not to be a substrate of p-glycoprotein (PGP, the relevant points are shown in red. WLOGP is the method for evaluating the lipophilicity of compounds shown in the ordinate diagram, and TPSA is the polar surface-area unit A of the compound.

Chemicals
All purchased reagents and raw materials were of analytical grade (AR) grade and were used directly without further purification. Nuclear magnetic-resonance (NMR) spectroscopy was carried out on a Bruker AVANCEIII-400 and an AVANCEIII-500 NMR. Compounds were dissolved in DMSO-d 6 or CDCl 3 , tetramethylsilane (TMS) was used as internal control, chemical shifts (δ) were expressed in parts per million (ppm), coupling constants were expressed in Hertz (Hz), and multiplicity was described as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet (m), and broad (br). High-resolution mass spectra (HRMS) were recorded using MALDI-TOF-MS/MS (Agilent). Solvent peaks were used as reference values with CDCl 3 at 7.26 ppm for 1 H NMR and 77.16 ppm for 13 C NMR, with DMSO-d 6 at 2.50 and 3.33 ppm for 1 H NMR and 39.52 ppm for 13 C NMR. High-performance liquid-chromatography (HPLC) analysis of all final compounds was conducted on a Shimadzu 20AT Series HPLC with an ZORBAX Extend-C18 column (5 µm, 100Å, 4.6 × 250 mm, Agilent). The mobile phase was methanol-water, acetonitrile-water, methanol-water (0.05 mol/L NH 4 Ac), or acetonitrile-water (0.05 mol/L NH 4 Ac); the flow rate was 1 mL/min; and the detection wavelength (λ) was 254 nano. All final compounds for biological evaluation were analyzed to achieve a minimum of 95% purity. Compounds were isolated and purified by column chromatography using 200-300-mesh silica gel. The reaction process was monitored by thin-layer chromatography (TLC) using pre-coated silica-gel plates (GF254) with a thickness of 0.25 mm under a UV lamp at a wavelength of 254 nm. For details of Nuclear magnetic-resonance (NMR) and High-resolution mass spectra (HRMS) of our compounds, please download the supplementary materials.
1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-3-(2,2,2-trichloro-1-(pyrimidin-2-ylamino) ethyl)urea (1 The synthesis of compounds was reported earlier by our research group [19]. Taking compound 20 as an example, 1.2 equivalents of p-nitrophenyl chloroformate (3.98 g, 19.80 mmol) were added to phenylethylamine (2.00 g, 16.50 mmol), dichloromethane (20 mL) was used as a solvent, and an appropriate amount of triethylamine was added to neutralize the hydrochloric acid produced by the reaction, and the reaction solution was directly cooled at low temperature. After stir for 1 h, aqueous ammonia (2.50 mL, 16.25 mmol) was added dropwise to the trap, stirred for 3 h, and the intermediate was obtained by adding methanol to precipitate. The intermediate and chloral hydrate (16.54 g, 0.10 mol) were stirred overnight and reacted at 100 • C, and ethyl acetate (40 mL) was added to separate out a white solid. Then, thionyl chloride (1.50 mL, 20.17 mmol) was added to reflux for 2 h and stirred to remove excess chloride. After the sulfone, an intermediate substituted with a nucleophile was obtained, which was stirred with 4,6-dichloro-1,5diaminopyrimidine (3.00 g, 16.75 mmol) in dry THF (30 mL) at 50 • C for 6 h to obtain a crude product, which was separated by column chromatography to obtain the target compound 20 (4.43 g, 9.43 mmol) as a white solid.